Efficacy and safety of PD‐1 monoclonal antibody combined with interferon‐alpha 1b and anlotinib hydrochloride as the second‐line therapy in patients with unresectable advanced melanoma: A retrospective study

Abstract Background Immune‐checkpoint inhibitors are now used more commonly in combination than monotherapy as the first‐line choice in patients with unresectable advanced melanoma. Nevertheless, for cases that progressed after the initial combination therapy, the subsequent regimen option can be very difficult. Herein, we reported the efficacy and safety of a triple combination regimen in Chinese unresectable advanced melanoma patients who had poor responses to the first‐line immune therapy. Methods We reviewed the clinical profiles of patients diagnosed with stage IIIC‐IV melanoma between June 1, 2020, and September 30, 2023. The patients who failed the prior immune therapies and received anti‐PD‐1 mono antibody plus interferon(IFN)‐alpha 1b and anlotinib hydrochloride as the second‐line therapy were enrolled in the retrospective analysis. Additionally, we examined the exhaustion of T‐cells using mIHC staining in available tumor samples. Results Fifty‐five patients were included in this study. The median follow‐up period was 13.6 months. The objective response rate evaluated by the investigators was 9.1%(1CR, 4PR). The disease control rate was 47.3%. The median overall survival was 17.6 months, and the median progression‐free survival was 2.8 months. The adverse events rate of any grade was 100%. Grade 3 or 4 irAEs were observed in 29.1% of cases. Multiplex immunohistochemical staining revealed an increased trend of TIM3 expression on tumor‐infiltrating T cells in patients without objective response. Conclusion PD‐1 monoclonal antibody plus interferon‐alpha 1b plus anlotinib showed acceptable tolerability and anticancer benefits in Chinese metastatic melanoma patients as a second‐line therapy.

Although immune checkpoint inhibitors (ICIs) have revolutionized the systemic strategy of cancer management including melanoma, the treatment resistance remains a great challenge that impairs their anti-tumor efficacy.4][5][6] Previously, we observed a 32.8% objective response rate in a retrospective cohort of Chinese metastatic melanoma patients treated with PD-1 antibody plus IFN-α1b, 7 which is higher than the response rate for either of the two drugs reported in former literature in similar cohorts.Nevertheless, the development of treatment resistance is still not uncommon in patients receiving ICI-based combination therapies.Usually, the majority of refractory cases respond poorer on second-line regimens, especially for those who do not carry targetable mutant genes.
Anlotinib is a novel oral multi-targeting receptor tyrosine kinase inhibitor featured by selective inhibition of vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptors (PDGFR), and c-kit.It was approved by the China Food and Drug Administration (CFDA) to treat advanced solid tumors including nonsmall-cell lung cancer, soft tissue sarcoma, medullary thyroid carcinoma, etc.[10][11] Therefore, we added anlotinib as a third drug to the combination of PD-1 antibody and IFN-α1b in advanced melanoma patients refractory to initial immune therapy and documented the treatment response as well as the toxicity of this regimen.

| Patients and study design
Profiles of patients with stage IIIC-IV melanoma diagnosed in the Dermatology Department of Xijing Hospital from June 1, 2020 to September 30th, 2023 were reviewed.The inclusion criteria were: (i).unresectable advanced melanoma (stage IIIC-IV, AJCC 8th Edition) confirmed by histological examination.(ii).Patients failed the firstline immunotherapy (PD-1 monotherapy or in combination with IFN-α 1b).(iii).Patients receiving the triple combination of IFN-α 1b (300ug or 600ug, subcutaneous injection, every other day) plus anti-PD-1 antibody (Pembrolizumab 200 mg/Toripalimab 240 mg/Sintilimab 200 mg, intravenous injection, every 3 weeks) plus anrotinib hydrochloride (oral, 12 mg daily for 14 days, discontinued for 7 days, every 21 days) as the second-line therapy for a least 8 weeks.Patients with absent or incomplete clinical information were excluded.
The curative effect of solid tumors was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST V1.1).Toxicity and grade were determined by Common Terminology Criteria for Adverse Events (CTCAE) V.5.0.This retrospective study was conducted by the Declaration of Helsinki.The Internal Review Board at the Air Force Military Medical University approved the project and waived the need for patient-informed consent.

| Statistical analyses
Continuous variables were reported as medians (ranges), while classified variables were reported as absolute frequencies and percentages.Kaplan-Meier curve is used to describe the progression-free survival (PFS) and overall survival (OS).Univariate and multivariate Logistic regression analysis was used to analyze the factors related to objective response rate (ORR).Univariate and multivariate COX regression analysis was used to analyze the risk factors affecting PFS and OS.In the multivariate survival analysis, the variables (p-value <0.10) that were significantly correlated with OS or PFS in the univariate analysis were introduced.In this paper, SPSS26.0 statistical software is used for statistical analysis.When p value<0.05, the difference is considered to be statistically significant.

| Multiplex immunohistochemical (mIHC) staining and image analysis
Multiplex immunohistochemical (mIHC) staining was employed to detect the T-cell exhaustion status in the tumor microenvironment of patients with and advanced melanoma, anlotinib, IFN-α1b, PD-1, second-line therapy without treatment responses.CD8α-70306 (Cell Signaling Technology (CST), Boston, USA), TIM3T-45208 (CST), LAG3-15372 (CST), PD1-86163 (CST), and S100-GZ031129 (Gene Tech, Shanghai, China) were used to obtain multiplex immunofluorescent staining.Multispectral imaging was also performed to aid in this process.The process involved the sequential application of distinct primary antibodies, incubation of secondary antibodies coated with horseradish peroxidase, and amplification of the tyramide signal.Following each TSA procedure, the slides underwent microwave heat treatment.After all human antigens had been labeled, nuclei were stained with 4′-6′-diamidino-2-phenylindole (DAPI, SIGMA-ALDRICH).The Mantra System (PerkinElmer, Waltham, Massachusetts, US) was used to scan the stained slides to create multispectral images.This system records the fluorescent spectra at 20-nm wavelength intervals from 420 to 720 nm with the same exposure time; the scans are then combined to create a single stack image.The spectrum of autofluorescence of tissues and each fluorescein was extracted from images of single-stained and unstained sections, respectively.Moreover, the recovered images were utilized to create a spectrum library that the inForm image analysis software (PerkinElmer, Waltham, MA, USA) needed for multispectral unmixing.We were able to generate reconstructed images of sections that had been stripped of autofluorescence by using this spectrum library.

| T-cell exhaustion markers
To evaluate the impact of T-cell exhaustion on the efficacy, we obtained 4 tumor samples from the individuals with treatment responses (1CR, 3PR) and 4 matched samples from those without treatment responses (1SD, 3PD).All samples were taken before the triple combination treatment was given.mIHC examination indicated a higher expression of TIM3 in patients without ORR.On the other hand, the expression of PD-1, LAG-3, and CD8 were similar between the two groups (Figure 3).Statistical calculation was not performed due to the small sample size.

| DISCUSSION
Though the efficacy of immune-checkpoint inhibitors in advanced melanoma has been improved by the combination of a second drug in most cases, the late-line management for patients without treatment response on the initial treatment is a greater challenge.Novel strategies have been developed to overcome the resistance to ICIs by reprogramming the tumor microenvironment (e.g., combination of dual immune-checkpoint inhibitors, adoptive T cell therapy, oncolytic treatments, cancer vaccines, etc.), for cases harboring specific pathogenic mutations, the addition of target inhibitors may bring synergetic benefits (e.g., BRAF and MEK inhibitors, PI3K inhibitors). 2,12,13In the current study, we performed a novel mixed regimen by adding anlotinib hydrochloride to a dual combination therapy of PD-1 and IFN-α1b which had previously been proven to have synergic efficacy in Chinese unresectable advanced melanoma patients. 14here were only five patients in the studied cohort who showed objective response on the triple combination regimen, yet 21 patients met stable disease criteria by RECIST V1.1, making the disease control rate 47.3% and mOS 17.6 months.Collectively, the triple combination of anti-PD-1 mono-antibody plus IFNα1b and anlotinib hydrochloride showed some efficacy but no significant survival advantage than those reported in other late-line regimens, such as Ipilimumab + Nivolumab, 15,16 lifileucel, 17 apatinib 18 and ceralasertib+ durvalumab (AZD6738), 19 in advanced melanoma patients who had failed the prior immune therapies.It needs to be pointed out clearly that many characteristics may affect the results in the current studied cohort, including but not limited to the subtypes, stage distribution, driver mutation percentage, and the front-line treatments.A majority of patients with BRAF mutation who failed the initial immune therapy in our center were prescribed dabrafenib + trametinib as the second-line medication, making the extraordinarily low portion of BRAF mutation in our cohort.
Treatment-related adverse events of any grade were reported in all patients (100%), but most of them were welltolerant, only 29.1% were grade 3/4 according to the CTC AE 5.0 definition.The most common events were fatigue, fever, and rash (all over 50%).Notably, oral ulceration was associated with ORR, and the rash was identified as  a predictor of longer OS, suggesting a correlation between cutaneous irAE and better treatment efficacy.
To determine the biomarkers predicting the treatment response, we performed mIHC staining in available tumor samples from the cohort of patients.Among the 4 markers we examined, TIM3 is the only one that showed an upward trend in patients who did not respond to treatment compared to those who had responses.1][22][23][24][25] However, we could hardly establish the correlation between TIM3 and the response of anlotinib addition due to the lack of control groups.In other words, the difference in TIM3 expression may reflect merely the response of the PD1 + IFN-α1b combination as the second-line therapy.However, these findings may suggest a reasonable use of TIM3 inhibitor as a late-line option after the failure of PD-1 + IFN-α1b.
The limitations of this study include the small sample size and potential distribution bias due to the retrospective cohort from a single center.Also, the nature of the retrospective study posed great difficulty in sample collections, obstructing us from response-related biomarker detection.Notably, excluding patients who received the combination therapy for less than 8 weeks may lead to potential selection bias.Moreover, the fact of sticking to the front-line immune therapies may have largely excluded patients who may develop severe adverse events in response to the triple combination therapy.Some of the issues above will be addressed in our prospective trial to assess the efficacy and safety of the triple combination of IFN-α1b + PD-1 monoclonal antibody + anlotinib as the first-line therapy in unresectable advanced melanoma(NCT05539118).

| CONCLUSION
The triple combination of PD-1 monoclonal antibody plus IFN-α1b and anlotinib showed acceptable toxicity and certain anticancer efficacy as the second-line therapy in Chinese advanced melanoma patients failed the prior immune therapies.

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55), and IV M1d was the least with only 3.6% (2/55).The acral subtype made up nearly a half of the enrolled cases (n = 27/55, 49.1%), the cutaneous subtype came second (n = 16/55, 29.1%) and the mucosal subtype was seen in 11/55 (20.0%).Twenty-six patients (47.3%) had elevated LDH.At the initiation of the second-line therapy, among the 55 patients, 53 had progressive disease, and 2 had stable disease in response to the front-line treatment.As to the best response to the first-line treatment of this cohort, 2 (3.6%) patients showed partial responses, 12 (21.8%)had stable disease, and 41 patients (74.5%

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I G U R E 3 (A-D)Tumor tissue from patients with objective response to triple therapy for mIHC.(E-H) Tumor tissue from patients without objective response to triple therapy for mIHC.R, objective response; NR, no objective response.
T A B L E 1 Univariable and multivariable Cox regression for OS.Summary of treatment-related irAEs.